PROJECT SUMMARY Transmembrane inner ear-expressed gene (TMIE) encodes a transmembrane protein that essential for hearing and vestibular function in vertebrates. A recent study of Tmie-/- mice has provided strong evidence for a central role of TMIE in mechanotransduction in cochlear hair cells. As a member of the transduction complex, mouse TMIE can bind to a specific isoform of Protocaderin 15 and/or another membrane protein, protein Lipoma HMGIC Fusion Partner-Like 5 (LHFPL5). Despite these seminal findings, it is not well understood how TMIE functions as a member of the transduction complex, and to date, its role in vestibular hair cells is unexplored. The aim of the present proposal is to conduct a comprehensive study of zebrafish Tmie in terms of identifying the protein motifs that are required for (i) localization of Tmie to stereocilia, (ii) modulation of mechanotransduction, and (iii) genetic and biochemical interactions with other members of the transduction complex. The proposed experiments will take advantage of our collection of mechanotransduction mutants and tools we have developed in zebrafish to investigate the function of Tmie in hair cells. Our preliminary data indicate that zebrafish Tmie is required the localization of Transmembrane channel like 1 (Tmc1) and Tmc2b to the stereocilia of hair cells in the inner ear and lateral line organ. This observation is surprising in light of experiments with Myc tagged TMC2 in mouse outer hair cells, however, the results suggest that TMIE may play different roles in different cell types. The proposed experiments will expand upon these novel findings and identify the amino acid motifs in Tmie that promote interaction with Lhfpl5a and the localization of the Tmcs to the site of mechanotransduction in hair cells. This work will provide mechanistic insights into the role of Tmie in hair cells and provide a better understanding the molecular determinants of Tmie that are critical for vertebrate hearing and balance.